Update on Usage of Cyanobacteria for Water Treatment
Biodesalination: A Case Study for Applications of
Photosynthetic Bacteria in Water Treatment1[C]
Jaime M. Amezaga, Anna Amtmann*, Catherine A. Biggs, Tom Bond, Catherine J. Gandy,
Annegret Honsbein, Esther Karunakaran, Linda Lawton, Mary Ann Madsen, Konstantinos Minas,
and Michael R. Templeton
School of Civil Engineering and Geosciences, Newcastle University, Newcastle upon Tyne NE1 7RU,
United Kingdom (J.M.A., C.J.G.); Institute of Molecular, Cell and Systems Biology, College of Medical,
Veterinary and Life Sciences, University of Glasgow, Glasgow G12 8QQ, United Kingdom (A.A., A.H.,
M.A.M.); Department of Chemical and Biological Engineering, University of Sheffield, Sheffield S1 3JD,
United Kingdom (C.A.B., E.K.); Department of Civil and Environmental Engineering, Imperial College London,
London SW7 2AZ, United Kingdom (T.B., M.R.T.); and Institute for Innovation, Design and Sustainability,
Robert Gordon University, Aberdeen AB10 7AQ, United Kingdom (L.L., K.M.)
Shortage of freshwater is a serious problem in many regions worldwide, and is expected to become even more urgent over the next
decades as a result of increased demand for food production and adverse effects of climate change. Vast water resources in the
oceans can only be tapped into if sustainable, energy-efficient technologies for desalination are developed. Energization of
desalination by sunlight through photosynthetic organisms offers a potential opportunity to exploit biological processes for this
purpose. Cyanobacterial cultures in particular can generate a large biomass in brackish and seawater, thereby forming a low-salt
reservoir within the saline water. The latter could be used as an ion exchanger through manipulation of transport proteins in the cell
membrane. In this article, we use the example of biodesalination as a vehicle to review the availability of tools and methods for the
exploitation of cyanobacteria in water biotechnology. Issues discussed relate to strain selection, environmental factors, genetic
manipulation, ion transport, cell-water separation, process design, safety, and public acceptance.
Bacteria are commonly employed for the purification
of municipal and industrial wastewater but until now,
established water treatment technologies have not taken
advantage of photosynthetic bacteria (i.e. cyanobacteria).
The ability of cyanobacterial cultures to grow at high
cell densities with minimal nutritional requirements (e.g.
sunlight, carbon dioxide, and minerals) opens up many
future avenues for sustainable water treatment applications.
Water security is an urgent global issue, especially
because many regions of the world are experiencing,
or are predicted to experience, water shortage conditions: More than one in six people globally are
water stressed, in that they do not have access to safe
drinking water (United Nations, 2006). Ninety-seven
percent of the Earth’s water is in the oceans; consequently, there are many efforts to develop efficient
methods for converting saltwater into freshwater.
Various processes using synthetic membranes, such
as reverse osmosis, are successfully used for large-scale
1
This work was supported by the Engineering and Physical Sciences Research Council.
* Address correspondence to [email protected].
The author responsible for distribution of materials integral to the
findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is:
Anna Amtmann ([email protected]).
[C]
Some figures in this article are displayed in color online but in
black and white in the print edition.
www.plantphysiol.org/cgi/doi/10.1104/pp.113.233973
desalination. However, the high energy consumption of
these technologies has limited their application predominantly to countries with both relatively limited freshwater
resources and high availability of energy, for example, in
the form of oil reserves.
The development of an innovative, low-energy biological desalination process, using biological membranes
of cyanobacteria, would thus be both attractive and pertinent. The core of the proposed biodesalination process
(Fig. 1) is a low-salt biological reservoir within seawater
that can serve as an ion exchanger. Its development can
be separated into several complementary steps. The first
step comprises the selection of a cyanobacterial strain that
can be grown to high cell densities in seawater with
minimal requirement for energy sources other than those
that are naturally available. The environmental conditions during growth can be manipulated to enhance
natural extrusion of sodium (Na+) by cyanobacteria. In
the second step, cyanobacterial ion transport mechanisms
must be manipulated to generate cells in which sodium
export is replaced with intracellular sodium accumulation. This will involve inhibition of endogenous Na+ export and expression of synthetic molecular units that
facilitate light-driven sodium flux into the cells. A robust
control system built from biological switches will be required to achieve precisely timed expression of the saltaccumulating molecular units. The third step consists
of engineering efficient separation of the cyanobacterial
cells from the desalinated water, using knowledge of
physicochemical properties of the cell surface and their
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Amezaga et al.
Figure 1. Proposed usage of cyanobacterial cultures for water treatment. A, Hypothetical water treatment station. Situated in basins
next to the water source, sun-powered cell cultures remove unwanted elements from the water. The clean water is separated from the
cells for human uses. The produced biomass is available for other industries. The proposed biodesalination process is based on the
following steps. B, Photoautotrophic cells divide to generate high-density cultures. C, The combined cell volume is low in salt as a result
of transport proteins in the cell membrane that export sodium using photosynthetically generated energy. D, Through environmental and
genetic manipulation, salt export is inhibited and replaced with transport modules that accumulate salt inside the cells. This process is
again fueled by light energy. E, Manipulation of cell surface properties separates the salt-enriched cells from the desalinated water.
natural ability to produce extracellular polymeric substances (EPSs), which aid cell separation while preserving
cell integrity. The fourth step integrates the first three
steps into a manageable and scalable engineering process.
The fifth and final step assesses potential risks and public
acceptance issues linked to the new technology.
In this review, we outline the state of knowledge and
available technology for each of the steps, as well as
summarize the current knowledge gaps and technical
limitations in employing a large-scale water treatment
process using cyanobacteria. Before discussing these issues, we provide some background information on the
usage of cyanobacteria in biotechnology and the impact
of sodium on cellular functions of cyanobacteria. The
example of biodesalination provides a good vehicle to
discuss the suitability of photosynthetic bacteria for water
treatment more generally. The issues addressed in this
review are relevant for a wide range of biotechnological
applications of cyanobacteria, including bioremediation
and biodegradation as well as the generation of biofuels,
natural medicines, or cosmetics.
CYANOBACTERIA IN BIOTECHNOLOGY
Cyanobacteria are a phylum of photosynthetic,
oxygen-producing bacteria, with a long evolutionary
history (Altermann and Kazmierczak, 2003). Because of
the process of complementary chromatic adaptation
(Bennett and Bogorad, 1973), cyanobacteria can utilize a
wide spectrum of photosynthetically active radiation as
their primary source of energy. Their long evolutionary
history has allowed them to adapt to a wide range of
environmental conditions and to occupy a vast array of
ecological niches. Modest growth requirements combined
with high adaptability generate a potential for harmful
algal blooms, which have earned these organisms some
bad publicity. However, cyanobacteria have contributed
to human nutrition for millennia either directly as a food
source or indirectly through nitrogen fixation in rice
paddies (Thajussin and Subramanian, 2005; Landsberg,
2010). More recently, biotechnological applications of
cyanobacteria have allowed for their utilization as animal
feeds and human food supplements and as producers of
bioenergy, cosmetics, and anticancer and anti-HIV drugs
(Spolaore et al., 2006).
In the context of environmental cleanup, Oscillatoria
salina, Plectonema terebrans, and Aphanocapsa sp. have
been used successfully for the degradation of crude oil
in seawater (Raghukumar et al., 2001). The applicability
of cyanobacteria extends to the remediation of heavy
metals (e.g. cadmium by Tolypothric tenuis) or even the
reclamation of precious metals (e.g. gold by Plectonema
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Cyanobacteria for Biodesalination
boryanum; Inthorn et al., 1996; Lengke et al., 2006). An
indication for possible usage of cyanobacteria for desalination was the reclamation of saline soils in India and the
Soviet Union using endemic strains (Apte and Thomas,
1997; Singh and Dhar, 2010). Thus, removal of cyanobacterial mats formed after rainfall also removed salt
from the soil. Further investigation of Anabaena torulosa
(a brackish strain) and Anabaena sp. strain L-31 (a freshwater strain) demonstrated that 90% of the salt accumulated was bound to EPSs at the cell surface, whereas the
remainder was internalized and osmotically active. The
freshwater strain showed a higher net sodium uptake
than the brackish strain, probably because of the higher
sodium efflux capacity of the latter. Interestingly, the influx of sodium was diminished in both strains by alkaline
pH, the high amount of extracellular potassium, or the
presence of nitrates or ammonium (Apte and Thomas,
1983; Apte et al., 1987). These observations suggested that
environmental triggers could be used to alter the magnitudes of sodium influx and efflux through endogenous
transport systems.
SELECTION OF SUITABLE
CYANOBACTERIAL STRAINS
Strain selection for biotechnological applications needs
to be guided by the purpose and the environment of the
envisaged process. With respect to biodesalination, candidate strains should meet a few key criteria. The culture
should be fast-growing to allow for the generation of
high cell density within a short time, thereby generating
a large cumulative internal volume and a large total cell
surface. The strain should be able to grow over a wide
range of external salt concentrations; the cells should be
able to adjust osmotically and to effectively export Na+
during growth. To allow for cell separation from the
water and other posttreatment procedures, the cells
should preferably be unicellular, possess a cell wall and
EPS, and have the capacity to adjust their buoyancy
(e.g. through intracellular gas vesicles). Finally, to facilitate genetic manipulation, the cells should be amenable to transformation techniques and their genome
sequence should be known.
Based on these criteria and an initial screen carried out
in one of our laboratories (Fig. 2), two strains emerge as
attractive candidates for biodesalination: the freshwater
euryhaline Synechocystis sp. strain PCC 6803 (Richardson
et al., 1983) and the marine-euryhaline Synechococcus sp.
strain PCC 7002 (formally Agmenellum quadruplicatum PR-6;
Ludwig and Bryant, 2012). Both strains are unicellular,
are capable of axenic growth, and are easy to maintain
under laboratory conditions. The genomes of both organisms have been sequenced (Kazusa DNA Research Institute, 2013) and successful transformation with foreign
DNA has been reported (see below). A particular advantage of Synechococcus sp. strain PCC 7002 is its high
growth rate. Generation times of less than 3 h have been
reported, making this strain the fastest dividing cyanobacterium and one of the fastest growing photosynthetic
Figure 2. Prescreening of cyanobacterial cultures for strain selection.
The effects of different media and environmental conditions on the performance of cyanobacterial cultures can be tested under controlled conditions in the laboratory. [See online article for color version of this figure.]
organisms (Van Baalen et al., 1971). Both strains have
been used extensively as models for the study of photosynthesis. This research has already provided a wealth of
scientific knowledge, including information on physiological adaptations to salinity and other environmental
factors (Nakamura et al., 2000; Ludwig and Bryant, 2012).
MANIPULATION OF ENDOGENOUS SODIUM
TRANSPORT IN CYANOBACTERIA
Any usage of unicellular systems such as cyanobacteria for the removal of sodium (Na+) from seawater or
brackish water requires an understanding of the potential effects of Na+ on cellular functions, which in turn
depend on the Na+ concentration. Some Na+ is necessary
for nutrient uptake (e.g. Na+-dependent HCO32 transport), nitrate assimilation, nitrogen fixation, and photosynthesis (Apte and Thomas, 1983; Maeso et al., 1987;
Espie et al., 1988). Na+ is also required for cell division in
heterotrophic cyanobacteria and for pH homeostasis in
alkaline environments (Miller et al., 1984). Deleterious
effects become apparent when the intracellular sodium
concentration exceeds a certain level, including destabilization of the fatty acids in the cell membrane (Huflejt
et al., 1990), inhibition of electron transport between
H2O and PSII (Allakhverdiev and Murata, 2008), and a
complete halt of photoautotrophic growth (Bhargava
et al., 2003). The exact level at which Na+ becomes toxic
depends on both endogenous and environmental factors
and differs between strains.
Successful salt acclimation of cyanobacterial cells depends on ambient concentrations and length of the exposure (Marin et al., 2004; Hagemann, 2010). It is a
multistage process that includes the readjustment of
ionic and osmotic potentials as well as wider physiological changes. Turgor adjustment is one of the earliest
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Amezaga et al.
responses to salt stress (Blumwald et al., 1983). It involves
the biogenesis and accumulation of compatible solutes
such as glycosylglycerol and Suc (Porchia and Salerno,
1996; Engelbrecht et al., 1999). Moreover, increased
osmolyte uptake has been observed in some strains under
salt stress, and this uptake appeared to alleviate some of
the effects caused by salinity (Fulda et al., 1999). If salt
stress persists, ionic adjustment becomes increasingly important, in particular the active extrusion of Na+ through
Na+/H+ antiporters, as well as P-type Na+-ATPases (Marin
et al., 2004; Wiangnon et al., 2007).
Environmental manipulations can make use of factors
that directly or indirectly alter the metabolism of the organism. The primary metabolism of cyanobacteria is
largely based on photosynthesis and is hence strongly
regulated by light. By altering the photoperiod, light intensity, or wavelength, metabolic processes can be induced or inhibited literally by the flick of a switch. The
availability of carbon, nitrate, and phosphate also exerts
significant control over growth, metabolism, and energy
status. In particular, cotransport of bicarbonate, phosphate, and nitrate with Na+ (Shibata et al., 2002; Matsuda
et al., 2004; Baebprasert et al., 2011) opens opportunities
to use these macronutrients to modulate Na+ uptake
rates. Altering the cell’s energy status through metal deficiencies will affect active Na+ export from the cell, which
consumes a large proportion of the cell’s ATP. Magnesium in chlorophyll and iron in heme groups are essential
components of the photosystem and are hence required
for photosynthetic activity, whereas inorganic phosphate
is required for oxidative phosphorylation. Deficiency of
these elements is the most common reason for cultures
entering the stationary phase, and it can thus be expected
that cells lose their capacity to exclude Na+ toward the
end of the growth period. Furthermore, metabolic activity
is affected by changes in pH and temperature. A systematic assessment of the effects of individual factors, and
of their combinations, on Na+ transport in Synechococcus
sp. strain PCC 7002 and Synechocystis sp. strain PCC 6803
is now required to provide a set of environmental triggers
that can be used to alter Na+ exchange between the cells
and the surrounding water.
The two methods that are most commonly used for
transferring foreign genetic material into cyanobacteria
are natural transformation and conjugation. Several detailed reviews have been published on the genetic manipulation of cyanobacteria in general (Koksharova and
Wolk, 2002; Vioque, 2007; Heidorn et al., 2011; Wilde and
Dienst, 2011). Here we will only give a short overview
with emphasis on available tools for Synechococcus sp.
strain PCC 7002 and Synechocystis sp. strain PCC 6803.
sp. strain PCC 7002 (Stevens and Porter, 1980; Essich
et al., 1990; Frigaard et al., 2004) and Synechocystis sp.
strain PCC 6803 (Grigorieva and Shestakov, 1982; Barten
and Lill, 1995; Heidorn et al., 2011) are naturally transformable, although the process of DNA uptake is incompletely understood. Among other factors, type IV pili,
which are also responsible for cell mobility, are an important part of the natural competence of Synechocystis sp.
strain PCC 6803 (Yoshihara et al., 2001, 2002). Only
double-stranded DNA can be used for natural transformation, but it is converted into single-stranded DNA as it
passes through the cell envelope. Inside the cell, the
double-stranded state is restored during recombination
with the chromosomal or plasmid DNA of the host (Essich
et al., 1990; Barten and Lill, 1995). A calcium-dependent
nuclease, located in or on the plasma membrane, was
proposed to be responsible for the degradation of one
of the two strands during DNA uptake in Synechocystis sp.
strain PCC 6803 (Barten and Lill, 1995). In Synechocystis sp.
strain PCC 6803, no further fragmentation of extracellular
DNA incorporated into the cell in this manner was observed (Barten and Lill, 1995; Kufryk et al., 2002).
So-called integrative or suicide plasmids are used for
natural transformation in the laboratory. These plasmids
are able to replicate in Escherichia coli, which is used for
cloning of the gene before transfer to the cyanobacterial
host. They allow the researcher to position the gene of
interest between two flanking regions of DNA that are
homologous to sequences of the cyanobacterial genome,
the so-called neutral sites. Neutral sites are regions
whose deletion or interruption has produced no phenotypic effect under all growth conditions investigated thus
far. Neutral sites are generally found in silent or redundant genes as opposed to intergenic or 39-untranslated
regions, which can execute regulatory functions on gene
expression (Wilde and Dienst, 2011). In Synechocystis sp.
strain PCC 6803 and Synechococcus sp. strain PCC 7002,
integration of foreign DNA between the two flanking
homologous regions usually occurs by a double crossover event mediated by the highly efficient homologous
recombination system of these strains. The recombination efficiency depends on the length of the homologous
stretches. The optimal length is different for different
strains, but generally the longer the better (Labarre et al.,
1989; Heidorn et al., 2011; Xu et al., 2011). Covalently
closed or linearized plasmids as well as PCR products of
the region of interest can be used in natural transformation. For Synechocystis sp. strain PCC 6803, transformation with circular plasmid DNA was found to be
approximately 30% more efficient than transformation
with linearized plasmid DNA (Kufryk et al., 2002). For
Synechococcus sp. strain PCC 7002, the use of linear
fragments was recommended to achieve high transformation efficiency (Xu et al., 2011).
Natural Transformation
Conjugation
Natural transformation involves the spontaneous
uptake of DNA from the environment and subsequent
integration into the host genome. Both Synechococcus
DNA transfer by conjugation consists of plasmid
exchange between different bacteria. In the laboratory,
three strains are typically used for conjugation, also
GENETIC MANIPULATION OF CYANOBACTERIA
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Cyanobacteria for Biodesalination
called triparental mating: the host cyanobacterium and
two E. coli strains. One E. coli strain carries the vector
containing the gene of interest (cargo plasmid) and the
second E. coli strain carries the conjugal plasmid. If
additional helper plasmids are needed, they usually
join the cargo plasmid in the first E. coli strain. Mixing
of the two E. coli strains causes the conjugative plasmid
to transfer to the E. coli strain that carries the cargo and
helper plasmids. The latter is then competent to conjugate with the subsequently added cyanobacterial
strain and to transfer the cargo plasmid to the new
host (Vioque, 2007; Wilde and Dienst, 2011).
The cargo plasmids used for conjugation are vectors
capable of autonomous replication in the host cyanobacterium, as well as in E .coli, where the initial cloning takes
places. Two types of vectors can be distinguished. Shuttle
vectors are hybrids between a native cyanobacterial
plasmid and an E. coli plasmid and therefore carry two
different origins of replication, one that is specific for the
particular cyanobacterium and one that is specific for
E. coli. Broad host range vectors carry only one replicon,
which functions in many different bacterial hosts, including cyanobacteria and E. coli (Heidorn et al., 2011).
For conjugal transfer of both types of vectors, certain
additional genetic elements are essential. Most importantly, in the donor cell, a relaxase/nickase of the mobility gene family (mob genes) recognizes and cleaves a
specific site within an origin of transfer. The DNA strand
with the covalently bound relaxase protein is displaced
from the plasmid by an ongoing conjugative DNA replication process. Through interaction of the relaxase with
components of a multiprotein, membrane-associated
mating pair formation complex, a type IV secretion system (tra genes), it is transported to the recipient cell together with the attached DNA. In the recipient cell, the
relaxase catalyzes the ligation of the transported DNA to
reconstitute the conjugated plasmid (Smillie et al., 2010).
The origin of transfer is the only sequence required in cis
for a plasmid to be conjugally transmissible, which is
why both the shuttle vector and the broad host vectors
carry this DNA sequence.
Other Techniques for DNA Transfer
Protocols enabling DNA transfer through electroporation have been developed for Synechocystis sp. strain PCC
6803 (Marraccini et al., 1993; Zang et al., 2007), but cell
recovery after the procedure is slow and there are reports
that this technique increases mutation rates in some cyanobacteria (Bruns et al., 1989; Muhlenhoff and Chauvat,
1996). In the future, transfer of DNA through cyanobacterial viruses (cyanophages) could become an attractive alternative, although appropriate genetic tools for
transduction have not yet been published. However,
nonlytic cyanophages that infect marine Synechococcus sp.
and have their genome stably maintained within the host
have already been described (McDaniel et al., 2002).
Furthermore, it is known that some cyanophages have a
broad host range and can cross infect both Prochlorococcus
and closely related Synechococcus sp., which has been
implicated in horizontal gene transfer of photosynthesisrelated genes (Sullivan et al., 2003; Weigele et al., 2007).
Those types of phages have potential for the development of genetic tools.
Technique of Choice and Current Limitations
The technique of choice for the genetic manipulation of
Synechocystis sp. strain PCC 6803 and Synechococcus sp.
strain PCC 7002 will depend on how the foreign gene
information should be maintained in the host cyanobacterium. As mentioned above, the process of natural
transformation involves DNA linearization and conversion
to a single strand (Porter, 1986), which makes this technique unsuitable for genes on an autonomously replicating
plasmid. In this case, conjugation is the method of choice
because it ensures that a circular plasmid resides in the
host at the end of the transfer (Vioque, 2007). Integration
into the host genome by natural transformation is desirable when long-term inheritance is the goal. It also potentially reduces gene dose variation caused by copy
number variations of autonomously replicating plasmids.
The downside of incorporation of foreign DNA by homologous recombination into the genome is that cyanobacteria generally have multiple copies of the chromosome
(e.g. 12 in Synechocystis sp. strain PCC 6803), and heterozygous cells are thus created. Subsequent segregation over
several generations is needed to ensure that the foreign
DNA is present in all copies (Heidorn et al., 2011).
For applications beyond a laboratory setting, it is essential that marker genes (e.g. antibiotic resistance genes)
do not remain in the genome. To achieve marker-free
genomic mutations, counterselection procedures have
been developed for both Synechocystis sp. strain PCC 6803
and Synechococcus sp. strain PCC 7002. For Synechocystis
sp. strain PCC 6803, the process requires two transformation steps (Cheah et al., 2013). With the first transformation, a cassette containing two marker genes, a
kanamycin resistance gene for positive selection and the
toxic mRNA interferase (mazF) gene for negative selection,
is inserted into the genome via homologous recombination. mazF is under the control of a nickel-inducible promoter. Successfully transformed cells are selected on
nickel-free kanamycin-containing media and subjected
to a second round of transformation, in which the entire
cassette inserted by the first transformation is replaced
with the gene of interest. Subsequent counterselection is
performed on kanamycin-free, nickel-containing medium.
Cells that have not lost the marker gene cannot grow
because of the induction of mazF. A similar counterselection method for Synechocystis sp. strain PCC 6803
uses the Bacillus subtilis levan sucrase (sacB) gene as negative selection marker (Lagarde et al., 2000). The disadvantage of this counterselection system is the requirement
for a separate Glc-tolerant strain of Synechocystis sp. strain
PCC 6803 as the chassis. An alternative strategy for Synechocystis sp. strain PCC 6803 is based on the Flippase/
Flippase Recognition Target (FLP/FRT) recombinase
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Amezaga et al.
system from Saccharomyces cerevisiae rather than counterselection. As with the above-mentioned counterselection
methods, two transformation steps are needed (Tan et al.,
2013). A successful counterselection procedure for Synechococcus sp. strain PCC 7002 is based on acrylate
toxicity and requires only one transformation step
(Begemann et al., 2013). Deletion of the gene annotated
as acetyl-CoA ligase (acsA) through its replacement
with the DNA fragment of interest via homologous
recombination overcomes growth inhibition by acrylate. Thus, positive transformants are identified by their
ability to grow on selective medium containing acrylate. To achieve expression of multiple heterologous
genes, the acsA gene can be reinserted into the genome at a neutral site (e.g. a pseudogene annotated
as glycerol phosphate kinase; glpK). The organic acid
counterselection method is also potentially applicable
to Synechocystis sp. strain PCC 6803, because AcsA
activity also confers acrylate sensitivity to this strain.
A major problem for the genetic manipulation of cyanobacteria is their efficient system of restriction enzymes
that destroy foreign DNA introduced by any transformation technique. One way to prevent DNA fragmentation is to ensure that the introduced DNA sequence
contains no sites that are recognized by the endogenous
restriction system. However, target sites differ between
cyanobacterial species, which is one reason why a shuttle
or broad host vector that is maintained in one species
might be digested in another. A second approach is used
in conjugation, in which the helper plasmid can encode
methylases that protect against restriction enzymes commonly present in many cyanobacteria (Vioque, 2007).
In conclusion, methods for genetic manipulation of
cyanobacteria have been established, but the number of
available tools is still limited. For example, a set of two
integrative vectors exist for Synechococcus sp. strain PCC
7002 that recombine not with the chromosome but with
endogenous plasmids (Xu et al., 2011). Because those can
reach copy numbers of up to 50, high-level gene expression is achieved. This elegant solution is not yet available
for Synechocystis sp. strain PCC 6803. On the other hand,
autonomously replicating plasmids are still missing for
Synechococcus sp. strain PCC 7002, although a recently
developed broad host range vector is a potential candidate (Huang et al., 2010).
DESIGNING A SYNTHETIC BIODESALINATOR
Generation of a Salt-Free Biological Reservoir
The core of the proposed biodesalination process consists of the establishment of a salt-free (or low-salt) biological reservoir within seawater that can serve as an ion
exchanger. Most marine organisms already contain such
a reservoir because they actively exclude and remove salt
from their bodies. Cyanobacteria employ a range of Na+
export proteins in their cell membrane (Fig. 3), all of
which are energized by the chemical energy carrier ATP.
ATP powers Na+ export either directly through Na+pumping ATPases, or indirectly through H+-pumping
ATPases, which generate a proton motive force that
drives H+/Na+ antiport (Marin et al., 2004; Wiangnon
et al., 2007). The ATP requirement offers an opportunity
to halt Na+ export by depleting internal ATP stores
using the environmental manipulations detailed above
(e.g. omitting photosynthetically efficient wavelengths
from the light spectrum, depleting phosphate, altering
pH, or chelating Mg2+, Fe2+, or other essential metals).
Simply changing the growth system from an open
system to a closed system once the culture has achieved
high cell density may already rapidly deplete nutrient
supply and exhaust ATP reserves.
Designing Light-Powered Transport Modules
Once active Na+ export has come to a standstill, there
will be net Na+ influx into a cell until equilibrium with the
external medium is reached. Further extraction of Na+
from the medium will then require an energy source. To
prevent renewal of Na+ export, the energy-harvesting
system employed during this phase should not use ATP
as an intermediate. Good candidates for ATP-independent
light-powered biological batteries are halorhodopsin (Hr)
proteins. Hrs naturally occur in extremely salt-tolerant archaea (haloarchaea) and are membrane-integral proteins of
the rhodopsin superfamily that form a covalent bond with
the carotenoid-derived chromophore all-trans-retinal
(Schobert and Lanyi, 1982; Klare et al., 2008). Absorption of a photon with a defined optimal wavelength
induces trans-cis isomerization of retinal, which triggers
a catalytic photocycle of conformational changes in the
protein, resulting in the net import of one chloride per
photon into the cytoplasm. The turnover rates for lightactivated ion pumps such as Hr are in the millisecond
range (Kolbe et al., 2000; Chizhov and Engelhard, 2001;
Essen, 2002; Kouyama et al., 2010).
To date, several Hr proteins from different species have
been characterized (Klare et al., 2008; Fu et al., 2012). The
Hr from Natronomonas pharaonis (NpHr) has been cloned
and successfully expressed in heterologous systems such
as E. coli, mammalian cells, and Xenopus laevis oocytes
(Hohenfeld et al., 1999; Seki et al., 2007; Gradinaru et al.,
2008). Expression of NpHr in X. laevis oocytes resulted in
a light-dependent Cl2-inward current and consequently a
negative shift in the membrane potential (Seki et al.,
2007). The opportunity to artificially manipulate a cell’s
membrane potential through NpHr in conjunction with
light-activated cation channels (e.g. channel rhodopsin)
has been exploited in the field of optogenetics, achieving
control of action potentials in nerve cells with potential
medicinal applications (Fenno et al., 2011; Zhang et al.,
2011). We propose here that the negative membrane
potential generated by Hr could also be used to drive the
accumulation of positively charged substances in cells.
Thus, the expression of Hr could energize the uptake of
nutrients (e.g. Ca2+, Mg2+, K+, Fe2+), or toxic metals (e.g.
Cd2+, Ni2+), into either plants or microorganisms, for
biofortification and bioremediation, respectively.
Expression of Hr in a high-density cyanobacterial culture should remove both Cl2 and Na+ from surrounding
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Cyanobacteria for Biodesalination
Figure 3. Na+ transport and its energization in different phases of the proposed desalination process. In the culture growth
phase (left), the cells generate a low-salt reservoir inside the salty environment through active export of Na+ by endogenous
transport proteins (light gray circles) across the plasma membrane (PM). These are either directly fueled by ATP (Na+-ATPases)
or, in the case of Na+/H+ antiporters, exploit the pH gradient established by H+-ATPases (dark gray circle). Na+ export from the
cytoplasm (cyto) therefore relies on ATP and the proton motive force generated from light energy captured by photosystems
(green box) and chemiosmosis (ATP-synthase, gray knob) in the thylakoid membrane (TM). In the desalination phase (right), Na+
export is halted through inhibition of photosynthetic ATP production. Instead, light energy is used directly by halorhodopsin
(pink circle) to pump chloride into the cells. The resulting negative membrane potential (Vm) draws Na+ into the cell through
Na+-permeable channel proteins (gray box).
seawater and thus provide a means for biodesalination.
The observed Km values of Hrs for chloride uptake
(approximately 25 mM for chloride; Duschl et al., 1990)
are in an optimal range for this purpose. To increase
the speed of Na+ accumulation, the Na+ conductance
of the membrane might need to be enhanced by coexpression of Na+-permeable channels or carriers with Hr
(Fig. 3). Candidate proteins with different affinities and
gating characteristics can be found in bacteria (Koishi
et al., 2004), animals (Koopmann et al., 2006), and plants
(Xue et al., 2011). The resulting light-powered salt accumulator bypasses the endogenous energy metabolism
(photosynthesis and respiration) and should therefore
remain functional even when increasing intracellular Na+
levels inhibit other metabolic functions of the host. A living cell would thus be transformed into a synthetic cell.
Ensuring Function and Robustness of the
Synthetic Biodesalinator
Although technologies for environmental and genetic
manipulation of cyanobacteria are advancing fast and
are predicted to enable realization of the core synthetic
salt accumulator, several additional challenges remain
to be solved. First, only the protein part of Hr can be
heterologously expressed in cyanobacteria. The essential
all-trans-retinal is usually added as a supplement in the
laboratory, but this is not sustainable in a large-scale
process. Little is known about whether the enzymes that
produce all-trans-retinal from b-carotene are present in
cyanobacteria. However, cyanobacteria as photosynthetic organisms already produce a wealth of carotenoids for light harvesting and photoprotection (Takaichi
and Mochimaru, 2007); thus, engineering a synthetic
pathway for the final enzymatic steps should not prove
too difficult. Second, progressive accumulation of NaCl
in the cells not only requires rapid osmotic adjustment of
the cells (which most cyanobacteria are capable of), but
also threatens to lead to destabilization of membranes
and proteins. It is therefore important that the cyanobacterial strain is resistant to high salt concentrations
and that the heterologously expressed Hr and channel
proteins are derived from naturally salt-tolerant species.
Additional measures such as increasing the external
Ca2+ concentration and altering lipid composition of the
membrane should also be explored. Finally, even if the
biological materials are salt resistant, the biodesalination
process will need to be limited to a very narrow time
window situated between the end of the growth phase
and the cell-removal phase. It is therefore essential to
obtain control over the expression of introduced genes.
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Gaining Control over Gene Expression in Cyanobacteria
Control over gene expression is exerted through promoter regions in the DNA, usually located immediately
upstream of the gene, which are recognized by effectors
(initiating transcription) as well as other regulatory proteins that link transcriptional activity to endogenous and
environmental stimuli. Obtaining control over transgene
expression in cyanobacteria requires the identification
and isolation of promoters that are responsive to the
specific triggers that will be used in the biotechnological
process (environmental changes or supplements). For
example, in the envisaged biodesalination process, promoters that are specifically active in the early stationary
phase of the culture could be cloned into the expression
vectors to activate the transgenes after the initial growth
period. To ensure specificity and precise timing of gene
transcription, the suitability of any candidate promoter
as a biological switch needs to be tested in a range of
conditions and systems.
Promoter studies in cyanobacteria to date have primarily focused on characterizing native transcriptional
regulation in response to different environmental stimuli.
Traditionally, Synechocystis sp. strain PCC 6803 was
studied as a model for photosynthesis and circadian
rhythm and several light-responsive promoters were
identified, including the light-responsive (LR) promoter1
(Marraccini et al., 1993) and the promoter of preprotein
translocase subunit (secA; Mazouni et al., 1998), as well
as the light-repressible promoter of PSI reaction center
subunits (psaAB; Muramatsu and Hihara, 2006), and the
promoter of light-repressed protein A homolog (lrtA;
Imamura et al., 2004). More recently, cyanobacterial
studies have turned their focus to biotechnological applications and numerous heavy metal-inducible promoters have been characterized (Peca et al., 2008; Blasi
et al., 2012) as well as the copper-inducible promoter
of plastocyanin (petE; Briggs et al., 1990; Ghassemian
et al., 1994; Buikema and Haselkorn, 2001) and the
copper-repressible promoter of cytochrome c553 (petJ;
Ghassemian et al., 1994). Furthermore, promoters tightly
regulated by nutrient availability have been characterized, including the promoter of the sodium-dependent
bicarbonate transporter (sbtA) regulated by inorganic
carbon availability (Wang et al., 2004) and the promoter
of ferredoxin-nitrite reductase (nirA) regulated by nitrogen source (Ivanikova et al., 2005; Qi et al., 2005).
The majority of studies characterizing cyanobacterial
promoter activity have been performed in the native
organisms. This poses a problem for transgenic applications because of potential crosstalk and/or recombination; therefore, in biotechnology, native promoters
are generally avoided in favor of promoters from
closely related organisms. The most common method
of gene regulation in bacteria is the lactose operon
repressor-operator (lacI-lacO); however, although this
works well in some strains of cyanobacteria such as
Synechococcus sp. strain PCC 7942 (Clerico et al., 2007), it
is not suitable for others, including Synechocystis sp.
strain PCC 6803 (Huang et al., 2010). Other promoters
that are well characterized in E. coli such as the so-called
PL and PR promoters of bacteriophage l have also
shown poor functionality in cyanobacteria (Huang
et al., 2010; Huang and Lindblad, 2013).
A range of different vectors and reporters have been
used to test promoter activity in cyanobacteria (Marraccini
et al., 1993; Ivanikova et al., 2005; Peca et al., 2008; Huang
et al., 2010; Xu et al., 2011; Blasi et al., 2012). In an attempt
to standardize the characterization of promoter activity
for synthetic biology applications, a method was developed in E. coli whereby promoter activity could be measured relative to an in vivo reference promoter based on
the fluorescence intensities of GFP as a reporter (Kelly
et al., 2009). The method was further developed using a
broad host range vector derived from the so-called IncQ
plasmid, RSF1010, for promoter analysis in Synechocystis
sp. strain PCC 6803 (Huang et al., 2010). Because of the
nature of the vector, this method can be applied to a wide
range of organisms likely to include other cyanobacterial
species.
In summary, some promoters regulated by different
stimuli have been identified and characterized in cyanobacteria. For these to be suitable for biotechnological
applications, the activity of these promoters must be
characterized in nonnative settings, and standardized
methods for characterization in cyanobacteria have
been developed. At this stage, the availability of effective biological switches is still a bottleneck for usage of
cyanobacteria as a chassis in synthetic biology and for
biotechnological applications.
STRATEGIES FOR CELL-WATER SEPARATION
Once biodesalination has occurred, efficient cell-water
separation is the next step of the proposed process
(Fig. 1). The notion of microorganisms as independent
unicellular entities is continuously challenged by research
into microbial biofilms (O’Toole et al., 2000). Nevertheless, exploitation of photosynthetic microorganisms in
water treatment has focused predominantly on the use
of unicellular microbial suspensions (i.e. planktonic cells
or suspended multispecies microflocs). Although the
ease of growth and maintenance favor use of planktonic
cultures of cyanobacteria for biodesalination, the separation of such cells from the desalinated water during
downstream processing without affecting the integrity of the cells and inadvertent release of sodium
chloride back into the desalinated water will be an
economical and technical bottleneck in the biodesalination process, as seen from previous attempts
at water treatment using photosynthetic microorganisms (Uduman et al., 2010; Lam and Lee, 2012;
Olguín, 2012; Schlesinger et al., 2012). The difficulty
in separating planktonic cyanobacterial cells from
aqueous suspensions stems from the fact that cells
have similar densities to water, cells behave like
colloidal particles because of the cell dimensions (few
microns), and cells possess charged surfaces that
stabilize cell suspensions.
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Cyanobacteria for Biodesalination
Metal Salts for Coagulation
The removal of photosynthetic microorganisms, especially bloom-forming cyanobacterial strains such as
Microcystis aeruginosa and Nodularia sp., from water has
been studied in the context of water treatment processes.
Therefore, the cell-liquid separation techniques have borrowed heavily from wastewater treatment procedures,
although centrifugation and filtration are employed when
product quality, especially of high-value chemicals, is to
be ensured. Nevertheless, coagulation- and flocculationbased processes are considered to be more energy efficient
and cost-effective than centrifugation and filtration
(Uduman et al., 2010; Lam and Lee, 2012). Inorganic metal
salts such as aluminum sulfate (4.8 to 5.8 mg/L and 65 to
70 mg/L; Chow et al., 1999; Drikas et al., 2001, respectively), ferric chloride (30 mg/L; Chow et al., 1998), and
polyaluminum chloride (4 mg/L; Sun et al., 2013) are
effective at separating out up to 99% of cyanobacteria
from water. Aggregation of cells with addition of metal
salts is mediated by the neutralization of surface charges
(Lam and Lee, 2012). In these studies, the added coagulants did not affect the cell membrane integrity or cause
toxin release from the cells during flocculation. However,
extensive cell damage and release of intracellular components can occur during floc storage and recycling,
downstream of the flocculation process (Sun et al., 2013).
A related issue is that coagulation is normally operated at
an acidic pH during water treatment, which photosynthetic organisms may not tolerate (Kim et al., 2011a).
Polyionic Polymers for Coagulation
Formation of aggregates through the use of synthetic
and organic polymers (i.e. polymer bridging) has been
investigated as an alternative to the use of metal salts,
with some success. Synthetic cationic polymers such as
polyethylenimine (20 to 30 mg/L; Zeleznik et al., 2002;
Arrington et al., 2003), polyacrylamide (3 mg/L; Jancula
et al., 2011) and the polyacrylamide-based Praestol
(1 mg/L; Pushparaj et al., 1993) are able to flocculate
cyanobacterial cells with between 80% and 90% efficiency
of cell removal. Praestol did not affect cell membrane
integrity, but polyethylenimine was shown to increase
cell permeability. The effect of polyacrylamide on the cell
viability was not tested. In addition to synthetic polymers, organic flocculants such as clay and chitosan enhance the flocculation ability of cyanobacteria (Divakaran
and Sivasankara Pillai, 2002; Pan et al., 2006a, 2006b;
Verspagen et al., 2006; Zou et al., 2006; Liu et al., 2010).
Although no adverse effect on cell membrane integrity
has been demonstrated with the addition of chitosan, the
use of clay and chemically modified clay, especially
chitosan-modified kaolinite, results in widespread death
and lysis of cyanobacterial cells (Shao et al., 2012). Because the conditions during floc formation such as temperature, ionic strength of the suspension medium, pH,
strain type, and cell concentration differ between studies,
the efficiency of the polymers in cell removal cannot be
directly compared. Moreover, the efficiency of cell-liquid
separation using flocculation-based technologies is not
consistent. It depends to a great extent on the surface
characteristics of the suspended cells and the polymers
present in the environment. These can be either natural
organic matter or polymers produced by the cells during
growth (i.e.EPSs, also known as algogenic organic matter;
Henderson et al., 2010; Teixeira et al., 2010).
Microbial EPSs for Coagulation
Microbial EPSs are categorized in two separate fractions based on proximity to the cell surface. EPSs positioned near the cell surface by noncovalent interactions
are termed bound EPSs, and those that are secreted into
the culture medium are called free or released EPSs
(Eboigbodin and Biggs, 2008). The aggregation of cells
within a biofilm is known to be aided by the favorable
interactions between physicochemistry of the cell surface
and the EPS (Karunakaran and Biggs, 2011). However,
when using coagulants, especially polyvalent metal salts,
to induce aggregation, EPSs of Aphanothece halophytica
and Microcystis aeruginosa increase coagulant demand
(Takaara et al., 2007, 2010; Chen et al., 2009, 2010;
Henderson et al., 2010). On the other hand, the presence
of EPSs, without the use of metal salts, can induce aggregation. The bioflocculation of kaolinite using released
EPSs isolated from cultures of Phormidium sp., Anabaena
circularis, Lynbyga sp., and Microcoleus sp. was previously
reported (Levy et al. 1990, 1992; Chen et al., 2011). The
role of EPSs in flocculation was recently proposed for
Synechocystis sp. strain PCC 6803 (Jittawuttipoka et al.,
2013). Moreover, bioflocculant activity is not limited to
released cyanobacterial EPSs (Taniguchi et al., 2005; Kim
et al., 2011a; Nie et al., 2011). Interestingly, the bound
EPSs of cyanobacterial and heterotrophic cells have also
been indicated to aid flocculation. In species such as
Arthrospira plantensis, T. tenuis, and Desulfovibrio oxyclinae, autoflocculation is induced when the cells are
exposed to environmental stress (Sigalevich et al., 2000;
Silva and Silva, 2007; Markou et al., 2012). Acinetobacter
calcoaceticus, a water isolate, will not only autoflocculate
but will also enhance the flocculation ability of other
bacteria (Simões et al., 2008). In addition, the bioflocculation of algae using EPSs does not affect cell
membrane integrity (Lee et al., 2009; Kim et al., 2011a).
In conclusion, the harvesting of biomass without
affecting the integrity of the cells is an important
area of research within industrial biotechnology. Bioflocculation of cells is a balance between the physicochemical properties of the cell surface and EPSs, and
could be a preferable alternative to chemical coagulants. However, to facilitate biomass harvesting using
bioflocculants at the industrial scale, a rigorous study
of the cell surface characteristics and EPS production
of the cells under relevant operating conditions has to
be carried out, especially because the cell surface and
EPSs have been shown to be affected by the environmental conditions (Eboigbodin et al., 2006, 2007;
Mukherjee et al., 2012). Overall, there is an urgent need
for in-depth characterization of surface properties and of
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Amezaga et al.
EPSs in photosynthetic organisms so that suitable cellwater separation technologies can be developed.
DESIGN OF AN INTEGRATED PROCESS
The overall aims of municipal wastewater treatment
plants and water treatment plants are to protect public
health in a manner compatible with environmental, economic, social, and political concerns. Wastewater treatment commonly utilizes biological processes relying on
microorganisms to take up dissolved organic matter and
nutrients. These processes take advantage of the fact that
microorganisms are relatively easy to remove through
settling or filtration. Biological treatment technologies
deployed in wastewater treatment include the activated
sludge process (aerobic suspended growth), trickling filters (as well as other attached-growth biological filters),
and membrane bioreactors (membrane filtration combined with a suspended growth bioreactor). More advanced configurations of the activated sludge process,
incorporating aerobic and anoxic zones, can be operated
for nutrient removal. There are increasing regulatory
pressures, such as those in the EU urban wastewater
treatment directive (European Union, 1991), to limit nitrate and phosphate contents with the aim to protect
downstream aquatic ecosystems from eutrophication.
This is achieved through nitrification, denitrification, and
phosphate uptake by different communities of bacteria.
Other biological technologies used in wastewater treatment plants are aerobic lagoons and various suspendedand fixed-growth anaerobic processes (including a range
of anaerobic digester and anaerobic filter designs).
Reactors designed to promote viability, functionality,
and high concentrations of photosynthetic organisms
may differ significantly from those used in biological
wastewater treatment, even if both are based on principles of attached and suspended growth. Evidently, light
is a key parameter and reactors used to grow algae may
prove more suitable in this respect. Many photobioreactor designs are only used at the laboratory scale
and recent advances in light-emitting diode technologies
offer an opportunity to efficiently supply the requisite
wavelengths of light for photosynthesis. However, at full
scale, this becomes less feasible in terms of operational
and capital costs, with a key challenge of providing and
regulating light exposure to photosynthetic organisms.
Large-scale open lagoons are an appropriate system to
achieve this. In common with many engineered algal
cultures, these are more favorable in locations with yearround high solar radiation and temperature (Su et al.,
2011). Nonetheless, many design improvements are still
needed in order to improve robustness, reduce energy
consumption, and optimize growth conditions for largescale production of photoautotrophs. Providing a feed
with the appropriate nutrient profile and suitable temperature, and mitigating against interference from other
indigenous microorganisms are other key challenges
linked to a transition from growing photosynthetic organisms at the laboratory scale to the industrial scale. Of
the nutrients required for photoautotrophic growth,
carbon dioxide is considered as the most significant,
because of the high proportion (approximately 50% of
dry weight) of carbon in the biomass of photoautotrophic organisms (Kim et al., 2011b). Large-scale growth of
photoautotrophic organisms relies upon huge amounts
of carbon dioxide, which must be delivered in an energyefficient manner. At the laboratory scale, this can be
easily provided by sparging with air and/or carbon dioxide. Bubbleless gas-transfer membranes, widely used
in the food industry, show promise for larger-scale delivery of carbon dioxide (Kim et al., 2011b). Overall, in
order to achieve improved reactor design, it is critical to
better understand the kinetics of nutrient acquisition and
photon capture by relevant organisms, so that their
growth and rates of photosynthesis can be properly
controlled.
A crucial operational issue common to both wastewater treatment and growth of photosynthetic organisms is delivering a sustainable and cost-effective
disposal or reuse route for the large volumes of biomass
that will inevitably be produced. Promising avenues to
achieve this exist. Notably, these include anaerobic digestion, biofuel production, or utilization as a feed
substrate in aquaculture. With respect to biofuel production, genetically modified (GM) strains of Synechocystis sp. strain PCC 6803 have been grown that secrete
energy-rich fatty acids (Liu et al., 2011). Experience
from disposal of sewage sludge shows there are a
number of challenges that will need to be overcome
before reuse of waste biomass from photosynthetic organisms will become viable. These include effective
low-energy dewatering and complying with the relevant legislation for reuse and disposal of biosolids,
which is likely to be a particular issue for GM biomass.
The impact of residual salt on downstream reuse applications also requires consideration. Although anaerobically digesting biomass has the major benefit of
generating methane (a potential energy source), algal
sludge tends to be of relatively low biodegradability
and methane yield (Bond et al., 2012). In such situations,
pretreatment or hydrolysis, to increase biodegradability, and/or codigestion with a complementary
feed source are possible methods to improve digester
performance.
The design of clarifiers for effective separation of
photosynthetic organisms from water is another important issue to consider when moving from a laboratory scale to a full scale of operation. Sedimentation and
flotation are two economically viable cell-liquid separation techniques typically employed in water treatment plants. Both approaches require coagulation and
efficient floc formation to achieve high separation efficiencies. Sedimentation has the advantage of low capital
expenditure and low energy consumption during operation compared with flotation. However, the ability of
cyanobacteria such as Synechocystis sp. strain PCC 6803
and Synechococcus sp. strain PCC 7002 to rise to the
surface of an open container (Fig. 4) suggests that flotation strategies such as dissolved air flotation can help
achieve high separation efficiencies rapidly.
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Cyanobacteria for Biodesalination
ASSESSMENT OF RISK AND PUBLIC ACCEPTANCE
Any application of biodesalination technology has
numerous health and environmental protection issues
that must be addressed during the design, construction,
and operation of the facility (World Health Organization,
2007). In addition, the use of synthetic biological applications, particularly involving cyanobacteria with
its known toxicity risks (Hunter et al., 2012), brings with it
the risk of low social acceptance (Bubela et al., 2012).
Indeed, the general public has historically been skeptical
about adopting alternative water sources in general
(Dolnicar et al., 2010) and proposed schemes have even
been abandoned because of a lack of public acceptance
(Po et al., 2003; Hurlimann and McKay, 2007; Hurlimann
and Dolnicar, 2010). Much research has been undertaken
into public acceptance of recycled water, particularly in
countries such as Australia, where serious droughts, with
their accompanying severe water restrictions, have led to
the search for alternative water supplies. More recently,
researchers have begun to also investigate public acceptance of desalinated water and have discovered different
degrees of acceptance, for both recycled and desalinated
water, depending upon the particular use intended
(Hurlimann and Dolnicar, 2011). Greater acceptance of
desalinated water, as opposed to recycled water, has been
found for close-to-body uses, whereas recycled water is
preferred for uses not close to the body (e.g. irrigation or
industrial cooling; Dolnicar and Schäfer, 2009; Dolnicar
et al., 2011).
Factors such as education, age, knowledge, income,
and sex influence acceptance levels of recycled water
(Dolnicar and Schäfer, 2009). In general, the more formal
the education received by a person, the greater their
knowledge about recycled water and the higher the
probability that they will accept it (Sims and Baumann,
1974). Related to this factor, Baumann (1983) found that
the better educated respondents had a greater faith in
science and technology and therefore a higher acceptance.
Similarly, Marks (2006) argues that effective public consultation promotes greater trust in those responsible for
the assessment and management of risks, and Po et al.
(2003) ascribe the success of a number of water reuse
projects to a great emphasis on public involvement and
education. As far as desalination is concerned, it has been
noted that the knowledge level concerning the technology is relatively low (Dolnicar et al., 2011); thus,
increasing the public’s knowledge could increase acceptance levels. Dolnicar et al. (2010) looked specifically
at how the provision of information about alternative
water supplies affected public perception. They concluded that hesitance to embrace such water is primarily
driven by water quality concerns, but providing people
with basic information about recycled and desalinated
water increased their likelihood of using these alternative supplies.
In addition to the general skepticism over the use of
desalinated water, the use of synthetic biological applications in the field of biodesalination, particularly
those involving GM cyanobacteria with their inherent
Figure 4. Cell-water separation can take advantage of the ability of
cyanobacteria to float. Visual appearance of initially mixed cultures of
cyanobacteria strains Synechocystis sp. strain PCC 6803 (top) and
Synechococcus sp. strain PCC 7002 (bottom) left under ambient laboratory light (8 6 2 mM) for 24 h (n = 3). [See online article for color
version of this figure.]
risks (Henley et al., 2013), increases the danger of low
social acceptance. Historically, public opinion on what
may be viewed as the (re)design of nature and the
merging of biology with engineering has been negative
(Bubela et al., 2012). As with the introduction of
recycled and desalinated water, however, the provision of accurate information on the benefits and risks
of the technology in the early stages of any proposed
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Amezaga et al.
project is believed to be critical, particularly concerning the image portrayed by the news media, which can
have an adverse influence on acceptance levels (Bubela
et al., 2012). Notwithstanding this, Christoph et al.
(2008) concluded that educating consumers does not
necessarily result in greater support for genetic modification because increased knowledge does not automatically imply support.
Despite the importance of public opinion to the success of emerging technologies, there remains a paucity
of studies in the literature on public perceptions of
synthetic biology. The majority of research has been
undertaken on social acceptance of GM food products
(Costa-Font et al., 2008; Siegrist, 2008; Dannenberg,
2009). It is frequently argued that consumer rejection of
such foods is the result of their introduction without
any perceived benefits to consumers, together with the
portrayed risks of genetically modified organisms
(GMOs) to the environment (Frewer et al., 2004). Other
factors such as ethical and moral considerations and
trust in both the scientists conducting the research and
the regulatory system are also important determinants
of consumer acceptance or rejection of the technology
(Frewer, 2003; Siegrist, 2008). In a study by Magnusson
and Koivisto Hursti (2002), it was discovered that age
and sex, together with level of education, had an impact
on likely acceptance of GM foods, with males and
younger respondents generally being more positive.
Meanwhile, Prokop et al. (2013) discovered that disease
risk resulted in significantly more negative attitudes toward GM products. However, with current stringent
regulations governing the use of synthetic biological
applications, such concerns should be minimized, especially if the public is kept reliably informed from the
early stages of development.
One of the key considerations in the application of the
biodesalination technology concerns potential locations.
Issues of saline waters, and the requirement for desalination to augment supplies, are well known in the Gulf
States and South America, where conventional desalination plants already exist (Dawoud, 2005). Social acceptance of emerging technologies has been shown to
vary between countries. In particular, the experience of
serious drought and water restrictions in Australia has
led to less resistance to recycled or desalinated water in
recent years (Dolnicar and Schäfer, 2009,) suggesting
that public opinions are affected by personal experiences. Historically, developing countries were less opposed to the concept of genetic modification. However,
Frewer (2003) noted an increasing resistance to the introduction of GM foods in developing countries as a
result of activity of national government organizations
that oppose the implementation of genetic technologies in
agriculture. Meanwhile, studies in Germany (Christoph
et al., 2008) and Sweden (Magnusson and Koivistro
Hursti, 2002) found strong negative tendencies to the
acceptance of genetic modification, with the main concern being uncertainty about possible long-term effects
to the environment and human health. Acceptability was
greater toward applications involving nonfood products,
however, because they are seen to be more beneficial,
less risky, and ethically correct, a point also noted by
Sorgo et al. (2012).
A biodesalination process based on GM cyanobacteria will present multiple challenges from the point of
view of social and regulatory acceptance. It has clearly
more chance of success in countries in which desalination is already an accepted practice, and where GMOs
are not seen as a threat by both government and population. The process will have to ensure that it fulfills all
safety requirements for GMO approval. It will also have
to prove that there is no danger coming from the use of
cyanobacteria and to actively deal with potential negative perceptions as a result of toxin generation. Consequently, it seems advisable to explore initially combined
uses of the low salinity water and biomass in productive
systems designed for saline arid environments.
CONCLUSION AND OUTLOOK
This article examined, using the specific example of
biodesalination, the challenges and opportunities associated with applications of cyanobacteria in water
treatment, many of which are pertinent to other biotechnologies. The key part of the conceptualized biodesalination process is to employ a low-salt biological
reservoir within the cyanobacteria as an ion exchanger.
Uptake of salt into these reservoirs would then be mediated by genetic and/or environmental manipulation
of the cyanobacteria. As exemplified by Synechocystis sp.
strain PCC 6803 and Synechococcus sp. strain PCC
7002, cyanobacteria have a number of attributes that
make them attractive for such applications, because
they are fast-growing, tolerant of a range of salt concentrations, and amenable to genetic transformation.
Furthermore, because the primary metabolism of cyanobacteria is based upon photosynthesis, nutrient requirements are minimal and active salt export during growth is
powered by sunlight. Solar radiation can also be used to
energize subsequent salt accumulation through expression of retinal ion pumps such as Hr. Protocols for genetic
manipulation of cyanobacteria through natural transformation and conjugation have been developed. As is the
case in other biotechnological processes, biodesalination
requires efficient separation of cells from water. Coagulation is a suitable method, because this can remove up to
99% of cyanobacteria and chitosan flocculants have no
adverse impact on viability of cyanobacteria. The design
and operation of an integrated biodesalination process is
likely to build on knowledge of both algal bioreactors and
wastewater treatment processes.
Notwithstanding these opportunities, challenges need
to be overcome at each stage of the proposed biodesalination process. Further research is needed to elucidate the impact of environmental factors, including
pH, temperature, and nutrients, on salt transport in cyanobacteria. A major bottleneck for easy genetic manipulation is the limited availability of vector backbones
that enable flexible rearrangement of essential elements,
and of robust promoters that can operate as biological
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Cyanobacteria for Biodesalination
switches in nonnative settings. Furthermore, separation
of planktonic cyanobacteria from water is difficult because
of their low density and molecular size, and the presence
of EPSs can have contradictory effects on aggregation.
Consequently, to fully optimize separation, more work is
needed to characterize the surface properties of both cyanobacteria and EPSs. Finally, the use of synthetic biological applications to produce recycled water brings the
risk of low social acceptance, although this varies geographically and may increase with further education.
ACKNOWLEDGMENTS
The authors of this article joined forces to develop methods for biodesalination after a Sandpit event (Water For All Challenge, 2010) organized by the
Engineering and Physical Sciences Research Council.
Received December 10, 2013; accepted March 5, 2014; published March 7,
2014.
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